Interaction assays

HEK 293T cells were cotransfected with RFP-target and Merlin-NLuc plasmids, lysed in 0.5 ml of 20 mM tris-Cl (pH 7.4), 0.15 M NaCl [tris-buffered saline (TBS)] and 2 mM MgCl₂, 0.5% NP-40, 1× HALT protease inhibitor mix (Thermo Fisher Scientific, Waltham, MA), and 2.5 U benzonase (Sigma-Aldrich, St. Louis, MO). Lysates were diluted 1:2.5 with TBS (pH 7.4) and 0.05% Tween 20 (TBST), and then, NLuc activity was measured using Nano-Glo reagent (Promega, Madison, WI). RFP or GFP fusion proteins were immunoprecipitated using RFP-Trap_MA or GFP-Trap_MA (ChromoTek, Hauppauge, NY), recovered using a magnetic stand, and washed four times with TBST. The beads were transferred to the wells of a white 96-well plate. NLuc luciferase activity was measured on a FlexStation 3 (Molecular Devices, San Jose, CA). Immunoprecipitation-associated luciferase was normalized to the activity in the lysates. RFP-Trap_MA beads were recovered, rinsed in TBS, resuspended in 1× Instant-Bands loading dye (EZBiolab, Indianapolis, IN), boiled for 10 min, and run on a 4 to 20% tris-glycine SDS-PAGE gel.